METHOD FOR THE GENERATION OF MONOCLONAL ANTIBODIES DERIVED FROM HUMAN B CELLS
The present invention relates, in general, to human B cells, and, in particular to a method of immortalizing and cloning human B cells and to monoclonal antibodies derived therefrom. The invention further relates to methods of using the monoclonal antibodies for therapeutic and diagnostic purposes.
This application claims priority from U.S. Provisional Application No. 61/322,725, filed Apr. 9, 2010 and U.S. Provisional Application No. 61/322,821 filed Apr. 10, 2010, the entire contents of which are incorporated herein by reference.
This invention was made with government support under Grant No. AI067854-02 awarded by the National Institutes of Health. The government has certain rights in the invention.
TECHNICAL FIELDThe present invention relates, in general, to human B cells, and, in particular to a method of immortalizing and cloning human B cells and to monoclonal antibodies derived therefrom. The invention further relates to methods of using the monoclonal antibodies for therapeutic and diagnostic purposes.
BACKGROUNDThe isolation and characterization of monoclonal antibodies that neutralize, for example, a broad spectrum of HIV-1 isolates are important to the design of an effective HIV-1 vaccine. An efficient method of obtaining such antibodies has, however, been elusive.
Epstein Barr Virus (EBV) has been used to immortalize human B cells that produce neutralizing antibodies (Traggiai et al, Nat. Med. 10(8):871 (2004) Epub 2004 Jul. 11; Corti et al, PLoS ONE 5:e8805 (2010)). The efficiency of immortalization, however, can be low, as can the cloning efficiency of the immortalized B cells.
The present invention provides a method for the rapid and efficient expansion of clonal memory B cells, for the isolation of antibody variable heavy (VH) and variable light (VL) genes and for the capture and growth of B cells producing broadly neutralizing antibodies (e.g., broadly neutralizing anti-HIV-1 antibodies).
SUMMARY OF THE INVENTIONThe present invention relates to human B cells. More specifically, the invention relates to a method of immortalizing and cloning human B cells, to monoclonal antibodies derived from such cells, and to nucleic sequences encoding VH and VL chains of the monoclonal antibodies. The invention further relates to methods of using the monoclonal antibodies for therapeutic and diagnostic purposes.
Objects and advantages of the present invention will be clear from the description that follows.
The present invention relates, at least in part, to a method of producing immortalized B cells. The method can comprise transforming B cells using EBV using standard protocols. While the Example below involves the use of IgG+ memory B cells obtained from peripheral blood, the method can be applied to other B cell subsets from other tissues (for example, from mucosal or lymphoid tissue) and to cells of other isotypes (for example, but not limited to, IgM+ naive B cells or IgA-memory B cells). The present method couples CD40 ligation and EBV transformation with TLR-9 ligation. This approach makes possible the rapid and efficient screening of large numbers of B cells.
In accordance with the invention, B cells transformed with EBV can be expanded in culture and the culture supernatant screened for the presence of antibodies having a desired antigen specificity (e.g., the antigen specificity of the antibody can be directed against a pathogen (e.g., HIV-1 or other pathogen referenced in PCT/US09/63271), chemical or toxin). Immortalized clones of the antibody-producing B cells can then be isolated and further cultured under conditions such that the antibodies having the desired antigen specificity are expressed. The antibodies can be isolated from the culture medium using standard techniques. The B cells can be expanded monoclonally, oligoclonally or polyclonally by varying the cell density in the cultures (for example, from cell densities of 10 or less cells/well for a predominantly monoclonal expansion to 100 or more cells/well for polyclonal expansion). Advantageously, the cells are seeded at a density of <10 cells/well.
Nucleic acid sequences encoding the monoclonal antibody (or VH and/or VL chains thereof) can be isolated from the cloned B cells using standard techniques.
In accordance with the invention, host cells (e.g., 293T cells) can be transfected with a construct comprising the nucleic acid sequences encoding VH and/or VL chains of the monoclonal antibody of interest under conditions such that those sequences are expressed and antibodies having the desired specificity are produced. Thus produced antibodies can be isolated (e.g., from the host cell or from media in which the host cells are cultured) using standard techniques.
Preferred antibodies of the invention are derived from CH0219 and include 1-27-G2, 1-27-G11 and 1-19-F10 (see
The antibodies of the present invention can be used, for example, diagnostically or therapeutically. For example, the antibodies can be useful in the identification and/or purification (e.g., using affinity purification techniques) of an individual polypeptide or other antigen against which they are directed. The antibodies can also be employed as reagents in, for example, immunoassays, radioimmunoassay (RIA) or enzyme-linked immunosorbent assays (ELISA). The antibodies can be labeled with a detectable label (such as a radioisotope, a fluorescent molecule or an enzyme). Antibodies produced by the methods disclosed herein can be used for detecting pathogens, such as HIV-1.
The invention also includes pharmaceutical compositions comprising antibodies of the present invention and a carrier. In some embodiments, pharmaceutical compositions comprising transformed and/or activated B cells of the presently disclosed invention are provided. Pharmaceutical compositions can also contain a pharmaceutical acceptable carrier or adjuvant.
The antibodies of the present invention, or fragments (e.g., antigen binding fragments) thereof, can be used for the treatment of disease, for the prevention of disease and/or for the diagnosis of disease. In some embodiments, the monoclonal antibodies of the presently disclosed subject matter can be administered to a subject in need thereof, in a pharmaceutical composition or medicament as described above.
Suitable routes of administration will depend on the disease to be treated, prevented or diagnosed but can include IV, IM, intra-nasal or subcutaneous. Administration directly to mucosal tissues can also be effected, when appropriate.
Certain aspects of the present invention are described in greater detail in the Example that follows. In the study of the Example, CD40L-expressing L cells were used, however, other CD40L-expressing cells (e.g., CD40L-expressing 293T cells, CD4+ T cells, or macrophages, dendritic cells, FDCs, thymic epithelial and endothelial cells), anti-CD40 antibodies (Galibert et al, Eur. J. Immunol. 25(3):733-737 (1995), Saeland et al, J. Exp. Med. 178(1):113-120 (1993), Bamchereau and Rousset, Nature 353(6345):678-679 (1991)) or other means for inducing CD40 ligation can be used (e.g., CD40 agonists). While the strain of EBV used in the Example was B95-8, other strains of EBV can also be used, as can other viruses that mediate comparable effects (e.g., Herpesvirus papio for monkeys). The TLR ligand used in the Example was ODN2006 (Traggiai et al, Nat. med. 10(8):871 (2004)) but the invention includes the use of other TLR-ligands (e.g., LPS, R848). A Chk2 kinase inhibitor was also used in the Example. Other anti-apoptotic agents can also be employed (e.g., ATM inhibitor or any inhibitor of oncogenic stress or DNA damage response). It will be appreciated that the stimulation cocktail used gave better yields than other stimulation regimens.
As described in the Example below, IgG+ memory B cells from peripheral blood were enriched through a two-step process: (1) depletion of CD2+, CD14+, CD16+, CD235a+ and IgD+ cells through magnetic bead separation using a cocktail of PE-conjugated antibodies as primary antibodies and commercially available anti-PE microbeads as secondary antibodies; (2) enrichment of IgG+ cells through positive selection with an anti-IgG microbead-conjugated antibody applied on the negatively selected cell fraction. Cells were stimulated for 14 days. At the end of the stimulation, cells are assayed in bulk for RNA-extraction and/or preserved in RNAlater for later treatment. A determination was made of the cell dilution that resulted in a monoclonal expansion according to the single-hit model of Poisson distribution in repeated experiments on HIV-1 chronic and uninfected subjects. The statistical model was validated by sequencing the VH chains of 4 selected wells both from fresh and in RNAlater-treated stimulated cells.
The results are summarized in
The methods disclosed herein can be practiced in connection with human and non-human mammals (and, as appropriate, cells (e.g., B-cells) derived therefrom), including primates, rats, mice, guinea pigs, rabbits, hamsters, domestic animals (e.g., dogs and cats) and farm animals (e.g., cows, pigs, horses).
Certain aspects of the invention are described in greater detail in
Complete Medium (CM) for PBMC, EBV-B cells, J774A.1, and K6H6/B5 cell lines
RPMI 1640 (Invitrogen) supplemented with:
-
- 1) 15.2% heat-inactivated fetal calf serum (FCS)
- 2) 1% non-essential amino acids (NEAA)
- 3) 1 mM sodium pyruvate
- 4) 15 mM HEPES buffer, pH 7.3
- 5) 2 mM L-glutamine (Glu)
- 6) 100 U/ml penicillin G (Pen)
- 7) 100 μg/ml streptomycin (Strep)
Preparation:
-
- 1. Thaw a bottle of FCS (500 ml/bottle, −20° C.) and incubate at 56° C. for 30 min for heat inactivation (inactivation of c′)
- 2. Let bottle cool down and add 31 ml of Glu+Pen/Strep (100×, 100 ml/bottle, −20° C.)
- 3. Aliquot the FCS-antibiotics mixture into tubes at 50 ml/tube and freeze at −20° C.
- 4. Add 100 ml of FCS-antibiotics mixture to bottle of RPMI 1640 (500 ml/bottle, 4° C.)
- 5. Add 6.2 ml of HEPES buffer (100×, 100 ml/bottle, 4° C.) into the bottle
- 6. Add 6.2 ml of sodium pyruvate (100×, 100 ml/bottle, 4° C.)
- 7. Add 6.2 ml of NEAA (100×, 100 ml/bottle, 4° C.)
- 8. Filter the CM through a 0.22 μm filter unit.
- 9. Total CM: 618.6 ml
- Thaw frozen PBMCs as per standard operating procedure
- Resuspend PBMCsat 10×106 PBMC/ml in PBS/1% BSA and round up to the higher 100 μl. Keep cells at 4° C.
- Prepare a master mix of the following PE-conjugated antibodies at the following concentrations (determined by titration of each single lot):
- Anti-CD235a [0.08 μl/106 PBMCs]
- Anti-CD2 [5 μl/106 PBMCs]
- Anti-CD16 [5 μl/106 PBMCs]
- Anti-IgD [5 μl/106 PBMCs]
- Anti-CD14 [20 μl/106 PBMCs]
- Aliquot 100 μl cells in 5 ml round bottom FACS tubes and split equal volumes of master mix in each tube
- Mix and incubate 30 mins at 4° C.
- Add 2 ml/tube PBS/1% BSA
- Centrifuge 1500 rpm 5 mins
- Remove supernatant without disturbing the pellet
- Add 100 μl/tube PBS/1% BSA
- Add 200 μl/tube anti-PE Microbeads
- Mix and incubate at 4° C. for 15 mins
- Add 2 ml/tube PBS/1% BSA
- Centrifuge 300×g for 10 mins
- Remove supernatant without disturbing the pellet
- Tap well the tube to loosen up the pellet to prevent cell clumps. Add 1 ml PBS/1% BSA/tube and mix well. Visually check for cell clumps. In case plumps are present, break them by mixing with a P1000
- Transfer tubes into the AutoMACS chiller for 5 ml tubes
- Run DEPLETES program
- Collect the flow-through tubes
- Combine the cells together in a 15-ml conical tube(s)
- Centrifuge 300×g 10 mins
- Resuspend in 200 μl PBS/1% BSA.
- Add 20 μl anti-IgG Microbeads (for up to 107 cells)
- Mix well and incubate for 15 minutes at 4° C.
- Add 2 ml PBS/1% BSA
- Centrifuge 300×g 10 minutes
- Resuspend in 1 ml PBS/1% BSA
- Transfer tube in 15-ml tube chiller
- Run POSSEL_S program
- Collect the positively selected fraction
- Centrifuge 1500 rpm 5 mins
- Remove SN without touching the bottom
- Resuspend in complete medium
- Determine accurate cell count and volume
- Stimulate with EBV suspension (B95-8; 1 ml/100,000 B cells), ODN2006 (2.5 μg/ml) and chk2-inhibitor (5 μM) overnight
- Meanwhile, γ-irradiated (75 Gy) CD40L-transfected L cells are plated overnight into 96-well tissue culture plates at 5,000 cells/well
- After overnight incubation, check EBV-transformed cell viability
- Resuspend cells at 80 viable cells/ml in complete media containing ODN2006 and chk2-i and transfer 100 μl/well (i.e. 8 cells/well, which equals 1 activated cell/well for chronically HIV-1 infected subjects)
- Aliquot 100 μl/well of cell suspensions in 96-well plate containing 5000 CD40L L cells in 100 μl/well, one plate/cell concentration (total 6 plates, i.e. 3 plates/cell aliquot)
- Incubate at 37° C. for 14 days
For IgG Memory B Cells
-
- 1) On the day before EBV infection, prepare irradiated CD40L-L cells (7,500 cGray) and distribute the cells into 96-well plates (round-bottom) at 5,000 cells/well (100 μl/well).
- 2) Centrifuge memory B cells in complete medium (CM) at 1200 RPM (300 g) for 5 min at 4 ° C.
- 3) Resuspend the pellet in 1 ml of CM (1×105 cells/ml) containing ODN 2006 (5.0 μg/ml) and Chk2 inhibitor II (10 μM).
- 4) Add EBV suspension (B95-8 cell supernatant*, stored in a −80° C. freezer or a liquid N2 tank) at 1 ml/1×105 memory B cells.
- 5) The final concentrations of ODN 2006 and Chk2 inhibitor II are 2.5 μg/ml and 5 μM, respectively.
- 6) Incubate the cells overnight at 37° C. in a 5% CO2 incubator.
- 7) After incubation, count the viable cells and resuspend them at 80 viable cells/ml in CM containing 5 μg/ml PS2006 (ODN 2006) and 10 μM Chk2 inhibitor II.
- 8) Distribute the cells at 8 cells/well (100 μl/well) into the 96-well plates with the irradiated CD40L-L cells.
- 9) Incubate the cells at 37° C. in a 5% CO2 incubator.
- 10) On Day 7, take 90 μl of supernatant from each well and replace with 100 μl of fresh CM containing 2.5 μg/ml PS2006 (ODN 2006) and 5 μM Chk2 inhibitor II.
- 11) On Day 14, collect 100 μl of supernatant from each well for functional assays.
- 12) Collect additional 20 μl of supernatant from each well for binding assays.
- 13) Mix and collect 30 μl of the cells from each well for a storage in RNA Later.
- 14) Add 160 μl of fresh CM containing 2.5 μg/ml of ODN 2006 and 5 μM of Chk2 inhibitor II. At this time, ODN 2006 may not be necessary.
- B95-8 (marmoset B cell line; University of North Carolina-Chapel Hill)
- CD40L-L cells (mouse fibroblasts transfected with human CD40L)
- ODN 2006 (Invivogen; tlrl-hodnb-5)
- Chk2 inhibitor II (EMD/Calbiochem; 220486)
Freezing
In 96-well culture plate, have cells cultured in 100 μl/well supernatant (i.e. remove supernatant in excess, if necessary)
Add 150 μl/well on RNAlater and freeze at −80° C.
Thawing
-
- Keep PBS cold throughout the procedure
- Thaw 96-plate and keep it on ice
- Transfer cells from wells into RNase-free sterile eppendorf tube (300 μl)
- Add sterile PBS up to 1.5 ml to each tube
- Spin in microcentrifuge at 300×g for 10 minutes.
- Aspirate the buffer and resuspend in 210 μl PBS
- Take 10 μl, add 10 μl trypan blue and count at the hemocytometer
- Keep cells on ice and perform RNA extraction as soon as possible
Alternatively, cells can be single-cell sorted into PCR plates in wells containing mastermix using FSC and SSC gating (no PI)
Optimization of Conditions for EBV Transformation of B Cells—Experiment #5 (96-Well Format)I. Purpose:
To improve Epstein Barr virus (EBV) transformation efficiency and to obtain monoclonal EBV-B cell lines without limiting dilution methods
II. Samples:
Memory B cells isolated from frozen/thawed PBMC by using a customized Miltenyi kit
III. Methods:
Memory B cells were incubated with EBV suspension (B95-8; 1 ml/100,000 B cells) for infection in the presence of ODN2006 (2.5 μg/ml)+Chk2 inhibitor (2-arylbenzamidazole; 5 μM) or IL-2 (1,000 IU/ml)+R848 (2.5 μg/ml). After overnight incubation, the cells were resuspended in the media containing the same concentrations of the above drugs and distributed into 96-well plates (round bottom) at 30 cells/well. The cells were co-cultured with γ-irradiated feeder cells as indicated in the Table III. Two weeks after EBV-infection, each well was examined under a microscope and the number of wells containing a clump of live lymphoblast cell line (LCL) determined to estimate the overall transformation efficiency. In addition, levels of total IgG in the culture supernatants were measured using an IgG-specific immunoglobulin (Ig) ELISA to determine transformation efficiency of IgG-producing B cells.
IV. Purpose:
To improve Epstein Barr virus (EBV) transformation efficiency and to obtain monoclonal EBV-B cell lines without limiting dilution methods
V. Samples:
Memory B cells isolated from frozen/thawed PBMC by using a customized Miltenyi kit
VI. Methods:
Memory B cells were incubated with EBV suspension (B95-8; 1 ml/100,000 B cells) for infection in the presence of ODN2006 (2.5 μg/ml)+Chk2 inhibitor (2-arylbenzamidazole; 5 μM) or IL-2 (1,000 IU/ml)+R848 (2.5 μg/ml). After overnight incubation, the cells were resuspended in the media containing the same concentrations of the above drugs and distributed into 96-well plates (round bottom) at 10 or 30 cells/well. The cells were co-cultured with γ-irradiated feeder cells as indicated in the Table VI. Two weeks after EBV-infection, we examined each well under a microscope and determined the number of wells containing a clump of live lymphoblast cell line (LCL) to estimate the overall transformation efficiency. In addition, levels of total IgG in the culture supernatants were measured using an IgG-specific immunoglobulin (Ig) ELISA to determine transformation efficiency of IgG-producing B cells.
Experiment 8-30—Total IgG ELISA
Experiment 8-31—Total IgG ELISA
RESULTS
This method was used to isolate broadly neutralizing anti-HIV-1 monoclonal antibodies from a chronically HIV-1 infected subject. The subject was infected with a clade A HIV-1 virus and showed broad serum neutralization. Approximately 20,000,000 peripheral blood mononuclear cells (PBMCs) were collected and approximately 30,000 viable IgG+ memory B cells were obtained after enrichment and overnight EBV transformation. The cells were cultured at a density of 8 cells/well.
At the end of stimulation, the 3,600 cultures were screened for total IgG production using an ELISA assay, clade B transmitted founder gp140 Env63521 binding (ELISA) and neutralization of the difficult-to-neutralize HIV-1 CAP45 strain (tier 3, clade C; TZM-bl assay (Li et al, J. Virol. 80:11776-11790 (2006)). Aliquots of cells from each well were both (1) further expanded for supernatant collection and liquid nitrogen freezing, and (2) froze in RNAlater.
In this experiment, 1799/3600 cultures (50%) secreted IgG, as expected. Of them, 24 cultures bound the gp140 Env63521 (1.33%) and 26 clones neutralized >50% of HIV-1 CAP45 (range: 51.2-85.6%). Only 1 clone that bound gp140 Env 63521 also neutralized HIV-1 CAP45.
Interestingly, three clones that neutralized HIV-1 CAP45 were of the IgA and IgM isotype. This is in line with the notion that there was enrichment for IgG-secreting memory B cells (80 to 90% purity), rather than complete purification.
The VH and VL chains from bulk cells frozen at the day of harvest of the 20 best neutralizers were amplified and it was found that 37% were monoclonal and 52.6% were oligoclonal (2 or 3 functional sequences per culture). On supernatant from cultures expanded for an additional week, a check was made for breadth of neutralization (5 difficult-to-neutralize viruses, including CAP45, and SVA as negative control) and breadth of binding to transmitted founder envelopes (3 envelopes, including Env64521). It was found that 2 clones (1-27-G11 and 1-19-B7) neutralized >50% ¾ of the viruses. Clone 1-27-G11 had only 2 VH chains. VH and VL chains are being expressed in a transfection system to retrieve the monoclonal antibodies. No neutralizing clones bound to the transmitted envelopes whereas 9 of the 24 transmitted gp140 Env63521-binding clones previously identified (37.5%) also bound to Env1086C and Env00MSA
All documents and other information sources cited above are hereby incorporated in their entirety by reference.
Claims
1. A method of producing immortalized B cells comprising transforming B cells with Epstein Barr Virus (EBV) under conditions such that said immortalization is effected.
2. The method according to claim I wherein said B cells are IgG+ memory B cells.
3. The method according to claim 2 wherein said B cells are obtained from peripheral blood.
4. The method according to claim 1 wherein said B cells are obtained from peripheral blood, mucosal tissue or lymphoid tissue.
5. The method according to claim 1 wherein said B cells IgM+ or IgA-memory B cells.
6. The method according to claim 1 further comprising expanding said immortalized B cells in culture.
7. The method according to claim 6 further comprising isolating cells from said expanded population that produce antibodies having a desired antigen specificity.
8. The method according to claim 7 further comprising cloning said isolated cells and isolating from said cloned cells nucleic acid sequences encoding said antibodies, or the variable heavy or variable light chain thereof.
9. The method according to claim 6 wherein said cells are expanded monoclonally, oligoclonally or polyclonally.
10. The method according to claim 1 wherein said B cells are mammalian B cells.
11. The method according to claim 10 wherein said B cells are human B cells.
Type: Application
Filed: Apr 11, 2011
Publication Date: Jan 31, 2013
Inventors: Barton F. Haynes (Durham, NC), Mattia Bonsignori (Durham, NC), Kwan-Ki Hwang (Durham, NC), Hua-Xin Liao (Durham, NC), David C. Montefiori (Durham, NC), Micah A. Luftig (Durham, NC)
Application Number: 13/640,190
International Classification: C12N 15/86 (20060101);
